Monovalent Gb3-/Gb2-Derivatives Conjugated with a Phosphatidyl Residue: A Novel Class of Shiga Toxin-Neutralizing Agent

Abstract

Shiga toxin (Stx)-1 and Stx-2 are involved in the pathogenesis of hemolytic uremic syndrome (HUS) and severe systemic complications following enterohemorrhagic Escherichia coli (EHEC) infection in humans. [1][2][3][4] Stxs have an AB 5 structure, in which a single catalytic A subunit is associated with five identical B subunits. The B pentamer is responsible for the toxin binding to eukaryotic cell-surface glycolipid receptors such as galabiosyl (Gb 2 )-ceramide and globotriaosyl (Gb 3 )-ceramide. The susceptibility of target cells to Stxs is dependent on the presence of the functional glycolipid receptors. 5,6) After binding, Stxs are internalized into the cells, and then the A subunit exerts RNA N-glycosidase activity, resulting in inhibition of protein synthesis by catalytic inactivation of 28S rRNA. 7,8) Thus, the binding of Stxs to the receptor is the primary event causing Stx-mediated diseases.The binding of Stxs to the receptors is attributed to the interaction of the B pentamer with the saccharide moiety of the receptors. However, the Stxs do not strongly bind to the Gb 3 monomer.9,10) It has been shown that Gb 3 molecules are clustered and localized in lipid rafts on the cell membranes. 11)The clustering of Gb 3 molecules is important for strong binding to Stxs. 12) In light of these findings, many synthetic compounds mimicking the natural receptors have been investigated for eliminating Stxs from the intestine and/or neutralizing Stxs in the circulation, as a therapeutic strategy for protecting patients from serious Stx-mediated diseases. 9,10,[13][14][15][16][17][18][19][20][21][22] All of these previously reported compounds possess repeated Gb 3 mimic structures in a molecule with various backbones on expecting clustering effect of the saccharide moiety, although there was one exception of adamantyl-Gb 3 , 10) a Gb 3 monovalent derivative, that was speculated to assemble into small aggregates for activity.In the present study, we synthesized novel Gb 3 -and Gb 2 -conjugated derivatives with a phosphatidyl residue, a sugar unit monomer in the chemical structure, which would be expected to form liposomes and clusters of the sugar unit to neutralize Stx-1 and Stx-2. These compounds showed strong neutralizing activity against not only Stx-1 but also Stx-2. In the present paper, we propose monovalent Gb 3 -/Gb 2 -derivatives conjugated with phosphatidyl residue as a new type of Stx-neutralizing agent. MATERIALS AND METHODS CompoundsThe structures of the compounds used in this study are shown in Fig. 1. Gb 3 and Gb 2 conjugated with phosphatidylethanolamine dipalmitoyl (PEDP) residue are referred to as Gb 3 -PEDP and Gb 2 -PEDP, respectively. Gb 3 conjugated with dipalmitoyl phosphatidyl hexyl (DPPA) residue is referred to as Gb 3 -DPPA. These compounds were dissolved in dimethyl sulfoxide at a concentration of 2 mM, then diluted 10 times in phosphate-buffered saline (PBS, pH 7.4) and stored at 4°C. The stock solutions were diluted with culture medium just before use, and sonicated for 10 min in a water ...

Upregulation of Shiga Toxin Receptor CD77/Gb3 and Interleukin‐1β Expression in the Brain of EHEC Patients with Hemolytic Uremic Syndrome and Neurologic Symptoms

“…The authors hypothesized that excess amounts of Gb3 may reduce toxin delivery to susceptible tissues. And indeed, Neri et al (18) could demonstrate that monovalent Stx ligands of phosphatidylethanolamine dipalmitoyl-Gb3 (Gb3-PEDP) and galabiosyl (Gb2)-PEDP strongly neutralized the cytotoxicity of Stx in vitro.…”

Specific Egg Yolk Immunoglobulin as a New Preventive Approach for Shiga-Toxin-Mediated Diseases

“…The cytotoxicity assay was performed as described previously [17], [18]. Briefly, 5×10 3 /well of HeLa 229 cells (kindly provided by Dr. T. Yoshida, Aichi Medical University, Aichi Prefecture, Japan) were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Nissui Pharmaceutical) supplemented with 10% heat-inactivated fetal bovine serum (ICN Biomedicals, Aurora, OH, USA), 100 U/ml penicillin G and 0.1 mg/ml streptomycin with 96-well flat bottomed culture plates (Costar; Corning Inc., Corning, NY, USA).…”

“…In contrast, it has been shown that the affinity of Stx-1 for the glycolipid receptor is higher than that of Stx-2 [22], [23]. Therefore, the Gb 2 - or Gb 3 -conjugated compounds that have been reported as drug candidates show a good neutralizing activity against Stx-1, but are less active against Stx-2, although we recently reported that Gb 2 /Gb 3 -conjugated to phosphatidyl residues is relatively effective at neutralizing Stx-2 [18]. Importantly, it has been speculated that the low affinity of Stx-2 for the receptor might be involved in the higher toxicity in vivo [22].…”

“…Various synthetic compounds that mimic natural receptors, Gb 2 -Cer or Gb 3 -Cer, have been studied for eliminating Stxs from the intestine by binding to and/or neutralizing Stxs in the circulation as a therapeutic strategy for protecting patients from serious Stx-mediated diseases [12]–[18]. Clinically, Stx-2 is more potent in the development of Stx-mediated diseases than Stx-1 [19], [20].…”

Recombinant Shiga Toxin B Subunit Can Induce Neutralizing Immunoglobulin Y Antibody

“…Synthetic Gb 3 -conjugated compounds have been actively investigated for this purpose. [4][5][6] In another approach, monoclonal antibodies against Stx have been shown to neutralize Stx. [7][8][9][10] Stx released from EHEC in the intestine enters the bloodstream to reach the target organs.…”